By use of near-infrared (NIR) spectroscopy, simultaneous, multiple-con-stituent estimation of important bioprocess parameters can be obtained in a time frame (< 1 min assay) that was previously unattainable. Therefore, with NIR spectroscopy the opportunity exists to incorporate real-time chemical information into bioprocess monitoring or control strategies which will lead to significant bioproeess improvements. The NIR spectroscopic analysis of unmodified whole broth samples for acetate, ammonium, biomass, and glycerol is described for an industrial <i>Escherichia coil</i> fed-batch fermentation bioprocess. For acetate and glycerol, suitable results were obtained from multiple linear least-squares regression (MLR) analysis. A more sophisticated partial least-squares (PLS) regression analysis was necessary to adequately model ammonium and biomass. The respective prediction errors (1σ) of 0.7 g/L, 1.4 g/L, 0.7 g/L, and 7 mmol/L for acetate, biomass, glycerol, and ammonium compare well with the error of the wet chemical reference methods used to derive the calibration algorithms.

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